2, 5-diamino-3, 4-disubstituted-1, 6-diphenylhexane isosteres comprising benzamide, sulfonamide and anthranilamide subunits and methods of using same

ABSTRACT

The present invention provides 2,5-diamino-3,4-disubstituted-1,6-diphenylhexane (DAD) isosteres comprising benzamide, sulfonamide and anthranilamide subunits, a pharmaceutical composition comprising such compounds, a method of using such compounds to treat retroviral, specifically HIV and more specifically HIV-1 and HIV-2, infections in mammals, particularly humans, a method of synthesizing asymmetric DAD isosteres comprising benzamide, sulfonamide and anthranilamide subunits, and a method of using such compounds to assay new compounds for antiretroviral activity.

This application is a divisional of Ser. No. 08/359,612 filed Dec. 20,1994, now U.S. Pat. No. 5,728,718.

TECHNICAL FIELD OF THE INVENTION

This invention relates to2,5-diamino-3,4-disubstituted-1,6-diphenylhexane (DAD) isosterescomprising novel, nonpeptidic and achiral subunits and, moreparticularly, to DAD isosteres comprising benzamide, sulfonamide andanthranilamide subunits. This invention also relates to a pharmaceuticalcomposition comprising such compounds, a method of using such compoundsto treat retroviral infections in mammals, a method of using suchcompounds in antiretroviral activity assays, and a method ofsynthesizing asymmetric DAD isosteres comprising benzamide, sulfonamideand anthranilamide subunits.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is a fatal disease, reportedcases of which have increased dramatically within the past severalyears. Estimates of reported cases in the very near future also continueto rise dramatically. Consequently, there is a great need to developdrugs and vaccines to combat AIDS.

The AIDS virus was first identified in 1983. It has been known byseveral names and acronyms. It is the third known T-lymphocyte virus(HTLV-III), and it has the capacity to replicate within cells of theimmune system, causing profound cell destruction. The AIDS virus is aretrovirus, a virus that uses reverse transcriptase during replication.This particular retrovirus is also known as lymphadenopathy-associatedvirus (LAV), AIDS-related virus (ARV) and, most recently, as humanimmunodeficiency virus (HIV). Two distinct families of HIV have beendescribed to date, namely HIV-1 and HIV-2. The acronym HIV will be usedherein to refer to HIV viruses generically.

Specifically, HIV is known to exert a profound cytopathic effect on theCD4+ helper/inducer T-cells, thereby severely compromising the immunesystem. HIV infection also results in neurological deterioration and,ultimately, in the death of the infected individual.

The field of viral chemotherapeutics has developed in response to theneed for agents effective against retroviruses, in particular HIV. Thereare many ways in which an agent can exhibit anti-retroviral activity.For example, HIV requires at least four viral proteins for replication:reverse transcriptase (RT), protease (PR), transactivator protein (TAT),and regulator of virion-protein expression (REV). Accordingly, viralreplication theoretically could be inhibited through inhibition of anyone or all of the proteins involved in viral replication.

The PR processes polyprotein precursors into viral structural proteinsand replicative enzymes. This processing is essential for the assemblyand maturation of fully infectious virions. Accordingly, the design ofPR inhibitors is an important therapeutic goal in the treatment of AIDS.

Anti-retroviral agents, such as 3'-azido-2',3'-dideoxythymidine (AZT),2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyinosine (ddI) are known toinhibit RT. There also exist antiviral agents that inhibit TAT.

Nucleoside derivatives, such as AZT, are the only clinically activeagents that are currently available for antiviral therapy. Although veryuseful, the. utility of AZT and related compounds is limited by toxicityand insufficient therapeutic indices for fully adequate therapy.

Numerous classes of potent peptidic inhibitors of PR have been designedusing the natural cleavage site of the precursor polyproteins as astarting point. These inhibitors typically are peptide substrate analogsin which the scissile P₁ --P₁, amide bond has been replaced by anonhydrolyzable isostere with tetrahedral geometry (Moore et al.,Perspect. Drug Dis. Design, 1, 85 (1993); Tomasselli et al., Int. J.Chem. Biotechnology, 6 (1991); Huff, J. Med. Chem., 34, 2305 (1991);Norbeck et al., Ann. Reports Med. Chem., 26, 141 (1991); Meek, J. EnzymeInhibition, 6, 65 (1992)). Although these inhibitors are effective inpreventing the retroviral PR from functioning, the inhibitors sufferfrom some distinct disadvantages. Generally, peptidomimetics often makepoor drugs due to their potential adverse pharmacological properties,i.e., poor oral absorption, poor stability and rapid metabolism(Plattner et al., Drug Discovery Technologies, Clark et al., eds.,Ellish Horwood, Chichester, England (1990)). Furthermore, since theactive site of the PR is hindered, i.e., has reduced accessibility ascompared to the remainder of the PR, the ability of the inhibitors toaccess and bind in the active site of the PR is impaired, and those thatdo bind are generally poorly water-soluble, causing distinct problems indrug delivery.

The design of HIV-1 protease inhibitors based on the transition statemimetic concept has led to the generation of a variety of peptidederivatives highly active against viral replication in vitro (Ericksonet al., Science; 249, 527-533 (1990); Kramer et al., Science, 231,1580-1584 (1986); McQuade et al., Science, 247, 454-456 (1990); Meek etal., Nature (London), 343, 90-92 (1990); Roberts et al., Science, 248,358-361 (1990)). These active agents contain a non-hydrolyzable,dipeptide isostere such as hydroxyethylene (McQuade et al., supra; Meeket al., Nature (London), 343, 90-92 (1990); Vacca et al., J. Med. Chem.,34, 1225-1228 (1991)) or hydroxyethylamine (Rich et al., J. Med. Chem.,33, 1285-1288 (1990); Roberts et al., Science, 248, 358-361 (1990)) asan active moiety which mimics the putative transition state of theaspartic protease-catalyzed reaction. Twofold (C₂) symmetric inhibitorsof HIV protease represent another class of potent HIV proteaseinhibitors which were created by Erickson et al. on the basis of thethree-dimensional symmetry of the enzyme active site (Erickson et al.,supra). A-77003 and other compounds designed on the C₂ symmetry areundergoing clinical trials in humans (Kempf et al., Antimicrob. AgentsChemother., 35, 2209 (1991); Kempf et al., U.S. Pat. No. 5,142,056).However, peptidic compounds, such as those described by Kempf et al.,which contain valinyl subunits, could undergo racemization to inactiveenantiomers, i.e., enantiomers which do not demonstrate antiretroviralactivity, and would, therefore, be expected to be. of limited utility.

Recent studies, however, have revealed the emergence of mutant strainsof HIV in which the protease is resistant to the C₂ symmetric inhibitors(Otto et al., PNAS USA, 90, 7543 (1993); Ho et al., J. Virology, 68,2016-2020 (1994); Kaplan et al., PNAS USA, 91, 5597-5601 (1994)). In onestudy, the most abundant mutation found in response to A7703 was Arg toGln at position 8 (R8Q), which strongly affects the S₃ /S_(3') subsiteof the protease binding domain. Shortening the P₃ /P_(3') residues ofA-77003 results in inhibitors that are equipotent towards both wild-typeand R8Q mutant proteases (Majer et al., 13th American Peptide Symposium,Edmonton, Canada (1993)). Inhibitors have been truncated to P₂ /P_(2')without significant loss of activity (Lyle et al., J. Med. Chem., 34,1230 (1991); Bone et al., J. Am. Chem. Soc., 113, 9382 (1991)). Theseresults suggest that inhibitors can be truncated and yet maintain thecrucial interactions necessary for strong binding. The benefits of suchan approach include the elimination of two or more peptide bonds, thereduction of molecular weight, the diminishment of the potential forrecognition by degradative enzymes, and the improvement of activityagainst certain drug-resistant strains.

The use of HIV protease inhibitors in combination with agents that havedifferent antiretroviral mechanisms (e.g., AZT, ddI and ddT) also hasbeen described. For example, synergism against HIV-1 has been observedbetween certain C₂ symmetric HIV inhibitors and AZT (Kageyama et al.,Antimicrob. Agents Chemother., 36, 926-933 (1992)).

The usefulness of currently available HIV protease inhibitors in thetreatment of AIDS has been limited by relatively short plasma half-life,poor oral bioavailability, and the technical difficulty of scale-upsynthesis (Meek et al., J. Enzyme Inhibition, 6, 65-98 (1992)). Thereremains an urgent need, therefore, for retroviral protease inhibitorsthat do not suffer from the disadvantages of currently availableretroviral protease inhibitors as well as effective methods of treatingretroviral infection, in particular HIV infection, involving theadministration of novel antiretroviral agents alone and in combinationwith other antiretroviral therapies.

Accordingly, it is an object of the present invention to provideantiretroviral compounds, specifically retroviral protease inhibitors,that are resistant to viral and mammalian protease degradation andwhich, therefore, have improved plasma half-life and oralbioavailability. It is a related object of the present invention toprovide a method of treating retroviral, specifically HIV and morespecifically HIV-1 and HIV-2, infection in a mammal, specifically ahuman, involving the administration of one or more of the antiretroviralcompounds of the present invention alone or in combination with one ormore other, currently available, antiretroviral therapies. Accordingly,it is also an object of the present invention to provide pharmaceuticalcompositions comprising the antiretroviral compounds. Another object ofthe present invention is to provide a method of using such compounds toassay new compounds for antiretroviral activity and a method ofsynthesizing the present inventive asymmetric antiretroviral compoundsso as to enable scale-up synthesis. These and other objects andadvantages of the present invention, as well as additional inventivefeatures, will be apparent from the description of the inventionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides symmetric and asymmetric antiretroviralcompounds of formula: ##STR1## wherein the stereochemistry of each ofthe benzyl groups on the carbon atoms adjacent to the carbon atoms withthe Y and Y' substituents is R or S. Y and Y' are the same or differentand are R-hydroxyl, S-hydroxyl, R-amino, S-amino or hydrogen. X and X'are the same or different and are ##STR2## wherein n is 0 or 1. a-e arethe same or different and are hydrogen, hydroxyl, halogen, sulfhydryl,carboxyl, carboxamido, a substituted or unsubstituted amino, OR'", aC₁₋₆ substituted or unsubstituted straight or branched chain alkyl, a(CH₂)_(m) Z", an O(CH₂)_(m) Z", or an N(R) (CH₂)_(m) Z". R'" is a C₁₋₆substituted or unsubstituted straight or branched chain alkyl. Z" isphenyl, pyridinyl, morpholinyl, piperazinyl, indolyl, quinolinyl,isoquinolinyl, thiazolyl, benzimidazolyl, aminothiazolyl, piperidinyl oroxazolyl. Also provided is a pharmaceutical composition comprising oneor more of the above-described compounds alone or in combination withone or more other currently available antiretroviral compounds. A methodof treating a retroviral, specifically HIV and more specifically HIV-1and HIV-2, infection in a mammal, particularly a human, is furtherprovided wherein a compound as described above is administered alone orin combination with one or more other currently available antiretroviraltherapies. Also further provided are a method of using such compounds toassay new compounds for antiretroviral activity and a method ofsynthesizing asymmetric compounds as described above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides DAD isosteres with benzamide, sulfonamideand anthranilamide subunits. The compounds are antiretroviral proteaseinhibitors. In particular, the compounds inhibit the protease of HIV,more specifically the protease of HIV-1 and HIV-2. The compounds arecharacteristically different from currently available antiretroviralprotease inhibitors. Such differences include, among others, resistanceto mammalian and viral protease degradation, which is believed to be dueto structural differences, namely the novel, nonpeptidic and achiralbenzamide, sulfonamide and anthranilamide subunits that have beenintroduced into the DAD isosteres. Such differences, such as resistanceto-protease degradation, result in improved plasma half-life and oralbioavailability.

The compounds provided by the present invention have the formula:##STR3## wherein the stereochemistry of each of the benzyl groups on thecarbon atoms adjacent to the carbon atoms with the Y and Y' substituentsis R or S. Y and Y' are the same or different and are R-hydroxyl,S-hydroxyl, R-amino, S-amino or hydrogen. X and X' are the same ordifferent and are ##STR4## wherein n is 0 or 1. a-e are the same ordifferent and are hydrogen, hydroxyl, halogen, sulfhydryl, carboxyl,carboxamido, a substituted or unsubstituted amino, OR'", a C₁₋₆substituted or unsubstituted straight or branched chain alkyl, a(CH₂)_(m) Z", an O(CH₂)_(m) Z", or an N(R) (CH₂)_(m) Z". The substituenton the substituted amino is SO₂ (CH₂)_(p) R", CO(CH₂)_(p) R",COO(CH₂)_(p) R", CONR₁ (CH₂)_(p) R", wherein p=0-20. R₁ is a C₁₋₄substituted or unsubstituted straight or branched chain alkyl, whereinthe substituent on the substituted C₁₋₄ alkyl is hydroxyl, amino,carboxyl or carboxamido, and R" is hydrogen, hydroxyl, halogen, aminocarboxyl, carboxamido, phenyl, pyridinyl, morpholinyl, piperazinyl,indolyl, quinolinyl, isoquinolinyl, thiazolyl, benzimidazolyl,aminothiazolyl, piperidinyl, oxazolyl, cyclopentane, cyclohexane or aC₁₋₂₀ substituted or unsubstituted straight or branched chain alkyl,wherein the substituent on the substituted C₁₋₂₀ alkyl is hydroxyl,amino, carboxyl, phenyl, pyridinyl, carboxamido or OR'. R' is a C₁₋₆substituted or unsubstituted straight or branched chain alkyl, whereinthe substituent on the substituted C₁₋₆ alkyl is hydroxyl, amino,carboxyl or carboxamido. R'" is a C₁₋₆ substituted or unsubstitutedstraight or branched chain alkyl, wherein the substituent on thesubstituted C₁₋₆ alkyl is hydroxyl, amino, carboxyl or carboxamido, a(CH₂)_(q) Z', an O(CH₂)_(q) Z', or an N(R) (CH₂)_(q) Z', wherein q is aninteger of 0 to 4, R is hydrogen or a C₁₋₄ substituted or unsubstitutedstraight or branched chain alkyl, wherein the substituent on thesubstituted C₁₋₄ alkyl is hydroxyl, amino, carboxyl or carboxamido, andZ' is phenyl, pyridinyl, morpholinyl, piperazinyl, indolyl, quinolinyl,isoquinolinyl, thiazolyl, benzimidazolyl, aminothiazolyl, piperidinyl,cyclopentane, cyclohexane or oxazolyl. The substituent on thesubstituted C₁₋₆ alkyl at a-e is hydroxyl, amino, carboxyl, carboxamido,or OR"". R""is a C₁₋₆ substituted or unsubstituted straight or branchedchain alkyl, wherein the substituent on the substituted C₁₋₆ alkyl ishydroxyl, amino, carboxyl or carboxamido. For (CH₂)_(n) Z", O(CH₂)_(n)Z", and N(R) (CH₂)_(n) Z", n=0-4, R is hydrogen or a C₁₋₄ substituted orunsubstituted straight or branched chain alkyl, wherein the substituenton the substituted C₁₋₄ alkyl is hydroxyl, amino, carboxyl, orcarboxamido, and Z" is phenyl, pyridinyl, morpholinyl, piperazinyl,indolyl, quinolinyl, isoquinolinyl, thiazolyl, benzimidazolyl,aminothiazolyl, piperidinyl, cyclopentane, cyclohexane or oxazolyl. Forexample, pyridinyl can be 2-,3- or 4-pyridinyl, morpholinyl can be4-morpholinyl, indolyl can be 2-indolyl, quinolinyl can be 2-quinolinyl,isoquinolinyl can be 1,2,3,4-tetrahydroisoquinolin-2-yl, thiazolyl canbe 2- or 4-thiazoly, benzimidazolyl can be 2-benzimidazolyl, andaminothiazolyl can be 2-aminothiazol-4-yl. The ring substituted with a-ecan contain a nitrogen and, when the ring does contain a nitrogen, thesubstituent a, b, c, d or e at the position of the nitrogen does notexist.

Preferred compounds include the compound of the above formula, wherein Xand X' are the same, n is 0, Y is R-hydroxyl, Y' is S-hydroxyl, and aand c-e are hydrogen and b is hydroxyl, or a, d and e are hydrogen and band c are hydroxyl, or a, c and e are hydrogen and b and d are hydroxyl,or a, c and e are hydrogen, b is hydroxyl and d is methyl, or a, c and dare hydrogen, b is hydroxyl and e is methyl, or a, d and e are hydrogen,b is hydroxyl and c is methyl, or a and c-e are hydrogen and b is amino.Also preferred is a compound of the above formula, wherein X and X' arethe same, n is 0, Y is S-hydroxyl, Y' is hydrogen, a and c-e arehydrogen, and b is amino or hydroxyl. Another preferred compound is acompound of the above formula, wherein Y is R-hydroxyl, Y' isS-hydroxyl, n is 0, and X and X' are different, wherein, on X, b-e arehydrogen, and a is N(H)C(O)(O)CH₂ -2-pyridinyl, and on X', a and c-e arehydrogen and b is hydroxyl.

An especially preferred compound is a compound of formula: ##STR5##wherein V is carbon or nitrogen, and W is oxygen, nitrogen, (CH₂)_(r),wherein r=0-20, or a single covalent bond. Preferred compounds of thisformula include compounds in which Y is R-hydroxyl, Y' is S-hydroxyl, bis hydrogen, methyl, chloro or hydroxyl, W is oxygen and R" is CH₂-phenyl. Other preferred compounds of this formula include compounds inwhich Y is R-hydroxyl, Y' is S-hydroxyl, b is hydrogen, V is carbon, Wis oxygen or H₂ or does not exist, and R" is methyl, ethyl, hydroxyl orCH₂ 2-pyridinyl.

An especially preferred compound is 2S,3R,4S,5S!-2,5-bis ((N-N-(2-pyridinylmethyloxy)carbonyl!anthranyl)amino!-3,4-dihydroxy-1,6-diphenylhexane.Also referred are compounds of this formula, wherein Y is S-hydroxyl, Y'is hydrogen, b is hydrogen, V is carbon, W is oxygen, and R" is selectedfrom the group consisting of CH₂ -phenyl and CH₂ -2-pyridinyl. Anespecially preferred compound is 2S,3S,5S!-2,5-bis ((N-N-(2-pyridinylmethyloxy)carbonyl!anthranyl)amino!-3-hydroxy-1,6-diphenylhexane.

Accordingly, the present invention provides symmetric and asymmetric2,5-diamino-3,4-disubstituted-1,6-diphenylhexane (DAD) isosterescomprising benzamide, sulfonamide, and anthranilamide subunits. Theasymmetric compounds can be asymmetric with respect to X and X' or withrespect to the substituents a-e that are present on X and X', inparticular with respect to substituted amino groups, for example.Representative compounds are presented in Tables I-V. The substituentson these compounds, in particular on the substituted amino groups, maybe further modified as necessary to affect activity and ease thepreparation of a given pharmaceutical formulation, for example.

The compounds of the present invention may be synthesized by methodsknown to those of skill in the art. For example, DAD (Kempf et al., J.Org. Chem., 57, 5692-5700 (1992); Stuk et al., J. Org. Chem., 59,4040-4041 (1994)) can be reacted with suitably substituted acid or acidchloride in methylene chloride, toluene, preferably dimethyl-formamideat ambient temperature, i.e., room temperature. The acids also can becondensed with DAD using standard peptide coupling agents (Bodanszky etal., In The Practice of Peptide Synthesis, Springer-Verlag, New York,N.Y. (1984)). Suitable methods of synthesis are described in Examples1-4. The asymmetric compounds of the present invention also may besynthesized in accordance with the present inventive method as describedin Examples 5-6 and in Schemes V-VIII. The sulfonamides are prepared byreaction of a suitably substituted benzene sulfonic acid withDAD/Boc-DAD or other such intermediates, such as those used in thesynthesis of Diol-10, Diol-48, DD-5, ND-4 and DN-11. Alternativelysulfonamides are prepared by the reaction of a suitably substitutednitro benzene sulfonyl chloride with the above intermediates. The nitrosubstituent is manipulated to provide the desired amino analogs, whichin turn can be protected, i.e., acetylated, alkylated or converted intohydroxyl or other such derivative by standard procedures as needed.

Also provided by the present invention is a pharmaceutical compositioncomprising a retroviral proliferation-inhibiting, particularly a HIVproliferation-inhibiting and more particularly a HIV-1 and/or HIV-2proliferation-inhibiting, effective amount of one or more of a compoundas described above, alone or in combination with one or more of acurrently available anti-retroviral compound, such as AZT, ddI, ddC,D4T, lamivudine or 3TC, in a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well-known to those who areskilled in the art. The choice of carrier will be determined in part bythe particular composition, as well as by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the polymer-bound compositiondissolved in diluents, such as water or saline, (b) capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solids or granules, (c) suspensions in an appropriateliquid, and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible carriers. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to theactive ingredient, such carriers as are known in the art.

The compounds of the present invention, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Accordingly, the present invention also provides a method of treating aretroviral, particularly a HIV infection and more particularly a HIV-1or HIV-2 infection, in a mammal, particularly a human, wherein aretroviral proliferation-inhibiting amount of one or more of the presentinventive compounds, alone or in combination with one or more otherantiretroviral therapies or compounds, such as AZT, ddI, ddC, D4T,lamivudine or 3TC, is administered to a mammal infected with aretrovirus, particularly HIV and more particularly HIV-1 or HIV-2, theproliferation of which is inhibited by a retroviralproliferation-inhibiting amount of a present inventive compound. Thedose administered to an animal, particularly a human, in the context ofthe present invention should be sufficient to effect a therapeuticresponse in the animal over a reasonable time frame. The dose will bedetermined by the strength of the particular composition employed andthe condition of the animal, as well as the body weight of the animal tobe treated. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular composition. Whatconstitutes a retroviral proliferation-inhibiting amount, particularly aHIV proliferation-inhibiting amount, and more particularly a HIV-1 orHIV-2 proliferation-inhibiting amount, of one or more compounds of thepresent invention, alone or in combination with one or more othercurrently available antiretroviral compounds can be determined, in part,by use of one or more of the assays described herein. Similarly, whetheror not a given retrovirus is inhibited by a retroviralproliferation-inhibiting amount of a compound of the present inventioncan be determined through the use of one or more of the assays describedherein or in the scientific literature or as known to one of ordinaryskill in the art.

One skilled in the art will appreciate that suitable methods ofadministering the compounds and pharmaceutical compositions of thepresent invention to an animal are available, and, although more thanone route can be used to administer a particular composition, aparticular route can provide a more immediate and more effectivereaction than another route. One or more of the present inventivecompounds, alone or in combination with one or more other antiretroviraltherapies or compounds, can be administered to a mammal, in particular ahuman, as a prophylactic method to prevent retroviral, particularly HIVand more particularly HIV-1 or HIV-2, infection.

Also provided by the present invention is a method of synthesizing theasymmetric compounds of the present invention. For example, a solutionof benzyloxycarbonyl chloride, preferably 1M, in CH₂ Cl₂, is addedslowly, preferably at a rate of 1 ml/min, to a solution of DAD in asolvent, preferably methylene chloride (preferred ratio of 1 g:500 ml),containing a base, preferably diisopropyl ethylamine or triethylamine.The resulting product is filtered to remove insolubles, washedsuccessively with 1% KHSO₄ brine and dried, preferably over anhydrous K₂CO₃, to provide monobenzyloxycarbonyl protected DAD (MZ-DAD). MZ-DAD canthen be manipulated using standard reactions to provide asymmetricinhibitors. The synthesis of asymmetric compounds of the presentinvention is described in Schemes V-VIII, and also in Examples 5 and 6.The synthesis of symmetric compounds of the present invention isdescribed in Scheme I.

In addition, compounds of the present invention, such as diol-38 (TableIV, compound 27) and diol-48 (Table IV, compound 34), demonstrate goodfluorescence. The determination of which compounds of the presentinvention fluoresce can be done in accordance with methods well-known tothose of ordinary skill in the art. Such fluorescence can be used toassay for antiviral activity of newly discovered compounds. For example,a fluorescent compound of the present invention can be used as astandard in an antiviral activity assay. The fluorescence of thecompound can be measured in the absence and presence of a viral enzyme,such as a retroviral enzyme, in particular a retroviral protease, e.g.,an HIV protease, in vitro. A test compound can then be added to the testsystem comprising the fluorescent compound and the enzyme. The decreasedfluorescence of the fluorescent compound of the present invention in theabsence and presence of the test compound can be measured andquantitated as a measure of the antiviral activity of the test compound.

The following examples further illustrate the present invention, but donot limit the scope thereof.

EXAMPLE 1

This example describes the synthesis of DAD isosteres with benzamide andanthranilamide subunits.

Compounds 5-36 were synthesized as shown in Scheme I. The inhibitor coreunit 3 (2S,3R,4S,5S-2,5-diamino-1,6-diphenyl-3,4-hexanediol) wassynthesized by McMurray coupling of natural Boc-phenylalaninal (Kempf etal. (1992), supra). The 2,5-diamino compound 3 (X═OH) was condensed withsuitably substituted benzoic acid using the1-H-benzatriol-1-yl-1,1,3,3-tetramethyl-ammonium tetrafluoroborate(TBTU)/1-hydroxybenzotriazole (HOBt)/diisopropylethyl amine (DIPEA)method to provide compounds 7-22. The 2,5-diamino compound 3 (where, inScheme I, X═OH) was condensed with suitably substituted N-acylanthranilic acid using the TBTU/HOBt/DIPEA method to provide compounds26-34. The N-acyl anthranilamides were prepared by reaction ofanthranilic acid with corresponding acid chloride. Compound 29 wasprepared by hydrogenolysis of a compound wherein R₂ was OCH₂ -phenyl.Deshydroxy compounds were prepared from compound 4 (Kempf et al. (1993),supra). The condensation of the 2,5-diamino compound 3 with suitablysubstituted phenylacetic acid using this procedure provided compounds 5aand 5b. The ethers 18a and 19a were prepared by the reaction ofmethyl-3-hydroxybenzoate with corresponding halides in the presence ofNaH according to Scheme II. 3-hydroxy-5-methylbenzoic acid (14a) wasprepared from sodium acetopyruvate according to Scheme III (Turner etal., J. Org. Chem., 24, 1952 (1959)). 2-methyl-5-hydroxy-benzoic acid(15a) was prepared by a Diels-Alder condensation of 2-methylfuran andethyl propiolate in the presence of an aluminum chloride according toScheme IV (McCulloch et al., Can J. Chem., 49, 3152 (1971)). Thedeshydroxy compounds 24 and 25 were prepared by condensation of2S,3S,5S-2,5-diamino-1,6-diphenyl-3-hexanol (4, X═H; Kempf et al.,Recent Advances in the Chemistry of Anti-Infective Agents, Royal Societyof Chemistry, Bently et al., eds., pp. 297-313 (1993)) with suitablysubstituted benzoic acid using the TBTU/HOBt/DIPEA method (Scheme I,X═H). The structures of all compounds thus prepared were established byproton nuclear magnetic resonance (¹ H NMR) spectroscopy and massspectral (FAB and/or high resolution mass spectra (HRMS)) analysis. ¹ HNMR spectra were recorded on a Varian XL-200 and 500 MHz spectrometer;data were reported in δ ppm scale relative to TMS. FAB spectra and HRMSwere recorded on a VG ZAB-2F spectrometer (Manchester, England) and on aVG70-250 spectrometer, respectively. ##STR6##

Bisamides, such as (2S,3R,4S,5S)-2,5-bis((3-hydroxyphenyl)carbonyl)amino!-3,4-dihydroxy-1,6-diphenylhexane (7),were prepared as follows. A solution of 60 mg (0.43 mmol) of3-hydroxy-benzoic acid, 122 mg (0.38 mmol) of TBTU, 59 mg (0.43 mmol) ofHOBT, 174 μl of DIPEA, and 100 mg (0.19 mmol) of 2S,3R,4S,5S-2,5-diamino-3,4-dihydroxy-1,6-diphenyl-hexane (Kempf et al., J. Org.Chem., 57, 5692 (1992)) in 4 ml of dimethylformamide (DMF) was stirredat room temperature for 1.5 h, after which the reaction was quenchedwith a drop of 4-(2-aminoethyl)-morpholine. The solvent and volatileswere removed under reduced pressure, and the residue was taken up inethyl acetate and washed sequentially with 10% KHSO₄, water, and aqueousNaHCO₃, brine-dried and concentrated in vacuo. Crude product wascrystallized from methanol:water (1:10) as a white solid, yielding 80 mg(78%) (MS(FAB) m/z 541 (M+); ¹ H NMR: δ).

2-methyl-5-hydroxybenzoic acid (15a; Scheme III) was prepared asfollows. A solution of 2-methylfuran (6.56 g) in CH₂ Cl₂ (120 ml) wasadded, at about 20° C. to a mixture of ethyl propiolate (7.84 g) andanhydrous AlCl₃ (10.64 g) in CH₂ Cl₂. The reaction mixture was allowedto stand at room temperature for 30 min with occasional shaking and wasthen shaken vigorously with cold water. The organic layer was extractedwith 5% NaOH solution (3×10 ml), and the combined aqueous layer wasacidified and extracted with ethyl acetate (3×20 ml). The combinedorganic layer was washed with water, brine-dried and partitioned withaqueous NaHCO₃ (3×10 ml). The bicarbonate-soluble fraction, afterreacidification and extraction with ether, gave 15a (1.9 g) as acolorless solid; mp 181-183° (lit 185°) (McCulloch (1971), supra). ¹ HNMR (CD₃ OD): δ2.48 (s,3H), 6.88 (dd, J₁ -3 Hz, J₂ =8.2 Hz, 1H), 7.09(d, J=8 Hz, 1H) and 7.36 (d, J=2.8 Hz, 1H).

3-hydroxy-5-methyl-benzoic acid (14a, Scheme II) was prepared asfollows. A mixture of 321 g (1.78 mol) of ethyl sodiumacetopyruvate(Marvel et al., Org. Syn. 1944 Coll., I, 238 (1944)), 400 ml aceticacid, and 400 ml water was stirred for 2 h. During this time, the solidwas dissolved and the solution became gray. The content of the flask waspoured onto 1 kg of crushed ice and 150 ml of sulfuric acid. Theresulting solid was separated by filtration, washed with cold water, anddried to afford γ-lactone 14b (Scheme III, 195 g (86%), mp 89-90° C.).The γ-lactone 14b (200 g, 695 mmol) and MgO (120 g, 3 mol) were addedwith stirring to 1.5 l of water, previously warmed on a steam bath. Thereaction mixture became deep reddish orange in color and turned lightbrown in about 15 min. Stirring was continued on a steam bath for anadditional 30 min after the complete addition of solid. The magnesiumoxalate and excess MgO were removed by filtration and washed with warmwater. The filtrate was concentrated in vacuo to 200 ml, placed in anice bath and acidified with dilute HCl to provide3-hydroxy-5-methylbenzoic acid 14a (27 g (28%), recrystallized fromether, mp 205-207° C. (lit. 207-208° C.) (Turner et al. (1959), supra),¹ H NMR (CDCl₃): 0.32. (s,3H), 6.8 (m, 1H), 7.2 (m, 2H), 8.7 (m, 1H)).

3- 2-((methoxy)ethoxy)ethoxy!benzoic acid (18a, Scheme II) was preparedas follows. A mixture of 240 mg (1.6 mmol) of methyl-3-hydroxybenzoate,200 mg of anhydrous K₂ CO₃ and 0.27 ml (2 mmol) of1-bromo-2(2-methoxyethoxy)ethane in DMF was heated with stirring at 50°C. for 6 h. Then, the DMF was evaporated under reduced pressure and theresidue was extracted with ether. Methanol (5 ml) and NaOH (2 ml) wereadded to the methyl benzoate and stirred at room temperature for 4 h.The methanol was removed under reduced pressure, washed with ether (5ml), acidified and extracted with ethyl acetate (3×10 ml). The combinedethyl acetate layer was washed with brine, dried and evaporated toprovide 18a (310 mg, 79%) as a thick liquid; ¹ H NMR (CDCl₃): 3.4 (s,3H), 3.5 (M, 2H), 3.7 (m, 2H), 3.9 (m, 2H), 4.2 (m, 2H), 7.16 (m, 1H),7.36 (m, 1H), 7.62 (m, 1H) and 7.69 (m, 1H).

3- 2-(phenoxy)ethoxy!benzoic acid (19a, Scheme II) was prepared asfollows. To a stirred and cooled solution of triphenyl phosphene (1.9 g,7.2 mmol) in anhydrous methylene chloride (5 ml), under argon, asolution of bromine (0.7 ml) in CCl₄ (2 ml) was added slowly during 15min. The stirring was continued for 30 min, after which 20phenoxyethanol (1 g, 7.2 mmol) in CH₂ Cl₂ (2 ml) was added slowly. Thereaction mixture was allowed to warm to room temperature and leftovernight. The excess of solvents were stripped off and the residue wasrepeatedly extracted with petroleum ether. Evaporation of petroleumether gave 2-phenoxyethyl bromide (1.4 g, 95%); ¹ H NMR (CDCl₃): 3.66(m, 2H), 4.30 (m, 2H) and 6.9-7.4 (m, 5H).

2-phenoxyethyl bromide was converted to 3- 2-(phenoxy)ethoxy!benzoicacid (19b) by following an analogous procedure as described for compound18a; (M+H)⁺¹ =258; ¹ H NMR (CDCl₃): δ 4.3 (m, 4H), 6.9-7.0 (m, 6H),7.2-7.5 (m, 3H) and 12.5 (s, 1H).

EXAMPLE 2

This example describes the synthesis of 2S,3R,4S,5S!-2,5-bis ((N-N-(2-pyridinylmethyloxy)carbonyl!anthranyl)amino!-3,4-dihydroxy-1,6-diphenylhexane (Diol-48).

Diol-48 (compound 34) was synthesized according to the followingreaction scheme: ##STR7##

A solution of methyl anthranilate (9.06 g, 60 mmol) in methylenechloride (100 ml) containing triethylamine (11 ml) was added slowly (ata rate of 2 ml/min) to a stirred solution of triphosgene in methylenechloride (250 ml) (Pavel et al., J. Org. Chem., 59, 1937-1938 (1994)).After complete addition, the stirring was continued for an additional 2hr and volatiles were removed by evaporation under reduced pressure. Thesalts were separated by dissolving the reaction mixture in anhydrousether followed by filtration under anhydrous conditions. The resultingisocyanate was used in the next step without further purification.

A solution of methyl anthranilate isocyanate (1 g, 5.64 mmol) and2-pyridinylcarbinol (0.616 g, 0.545 ml, 5.64 mmol) in 40 ml of toluenewas stirred at room temperature under N₂ atmosphere for 36 hr. Thesolvent was removed under vacuum, and the residue was purified by ilicagel chromatography using the solvent system EtOAc-hexanes 1:1, to give0.72 g (45%) of the carbamate. MS(M+H)=287; ¹ H NMR (DMSO-d₆), δ 3.85(s, 3H), 5.25 (s, 2H), 7.1 (m, 1H), 7.3-7.4 (m, 2H), 7.6 (m, 1H),7.8-7.9 (m, 2H), 8.1 (m, 1H), 8.58 (m, 1H), 10.4 (s, 1H).

The resulting product was dissolved in dioxane (15 ml) and saponifiedwith lithium hydroxide (19 ml, 1N) at room temperature for 2 hr. Thevolatiles were removed, and the residue was acidified to pH 5.5 andextracted with ethyl acetate. Evaporation of the organic layer provided(N-2-pyridinylmethyloxycarbonyl)-anthranilic acid (Py-Ant).

A solution of methyl N 2-pyridinylmethoxy)carbonyl! anthranilic acid(56.5 mg, 0.2 mmol), DAD (30 mg, 0.1 mmol), HOBT (31 mg, 0.2 mmol), TBTU(65 mg, 0.2 mmmol), and DIPEA (52 mg, 70 μl, 0.4 mmol) in DMF (10 ml)was stirred at room temperature for 6 hr. The solvents were removedunder vacuum, and the residue was diluted with ethyl acetate, washedsequentially with a solution of KHSO₄ (pH=5), water, and aqueous NaHCO₃,and dried over MgSO₄. The yield was 54 mg of crude product. MS(M+H)=809; ¹ H NMR (DMSO-d₆), δ 6 2.80-2.85 (m, 1H), 2.89-2.93 (m, 1H),2.94-3.01(m, 2H), 3.59-3.66 (m, 2H), 4.57-4.67 (m, 2H), 5.10-5.20 (m,5H), 5.44(d, J=3.2Hz, 1H), 7.25-7.46 (m, 18H), 7.73-7.81(m, 4H),8.09-8.12 (m, 2H), 8.32 (d, J=3.6 Hz, 1H), 8.49 (d, J=3.6 Hz, 1H), 8.54(m, 2H), 10.60 (s, 1H), 10.76 (s, 1H). K_(i) =64 pM.

EXAMPLE 3

This example describes the synthesis of Diols 43, 39B, 38, 42, 36 and45.

Diol-43 (compound 32, Table IV) was prepared following a proceduresimilar to that described for Diol-48 (compound 34, Table IV), using 1.1molar equivalent of 3-chloro-N-((benzyloxy)carbonyl)-anthranilic acidinstead of N-((2-Pyridinylmethyloxy)carbonyl)-anthranilic acid.

Diol-39B (compound 29, Table IV) was prepared following a proceduresimilar to that described for Diol-48 (compound 34, Table IV) using 1.1molar equivalent of N-((2-benzyloxy)methyl)carbonyl!-anthranilic acidinstead of N-((2-Pyridinylmethyloxy)carbonyl)-anthranilic acid. Theintermediate Diol-39, a compound like compound 29 with the exceptionthat the R₂ group is OCH₂ -phenyl, thus obtained was debenzylated byhydrogenation with 10% Pd on carbon to provide Diol-39B. C₃₆ H₃₈ N₄ O₈ ;MW=654; MS (M+Na)+=677; ¹ H NMR (methanol-d₄), δ 2.82-2.98 (m, 2H),3.03-3.11 (m, 4H), 3.73 (s, 2H), 3.85 (m, 1H), 3.94-4.01 (m, 5H),4.65-4.75 (m, 2H), 7.08-7.51 (m, 18H), 8.31 (d, J=8 Hz, 2H).

Diol-42 (compound 31, Table IV) was prepared following a proceduresimilar to that described for Diol-48 (compound 34, Table IV), using 1.1molar equivalent of 3-methyl-N-((benzyloxy)carbonyl)-anthranilic acidinstead of N-((2-Pyridinylmethyloxy)carbonyl)-anthranilic acid. C₅₀ H₅₀N₄ O₈ ; MW=834; MS (M+Na)+=856; ¹ H NMR (CDCl₃ δ 2.15 (m, 6H), 2.85-2.89(m, 2H), 2.99 (m, 3H), 3.08 (dd, J=5.6; 1.8 Hz,2H), 3.66 (bs, 4H),4.57-4.64 (m, 2H), 4.96 (m, 2H), 6.9 (m, 1H), 6.96-7.02 (m, 2H),7.17-7.25 (m, 26H).

Diol-36 (compound 26, Table IV) was prepared following a proceduresimilar to that described for Diol-48 (compound 34, Table IV), using 1.1molar equivalent of N-acetyl-anthranilic acid instead ofN-((2-Pyridinylmethyloxy)carbonyl)-anthranilic acid. C₃₆ H₃₈ N₄ O₆ ; MS(M+H)⁺ =633; ¹ H NMR (methanol-d₄), δ 1.88 (s 3H), 2.01 (s, 3H), 2.83(m, 1H), 3.03-3.11 (m, 3H), 3.63 (s, 1H), 3.79 (m, 2H), 4.73 (m, 2H),7.06 (m, 1H), 7.14-7.45 (m, 14H), 7.54 (m, 1H), 8.07 (m, 2H).

Diol-45 (compound 30, Table. IV), was prepared following a proceduresimilar to that described for Diol-48 (compound 34, Table IV), using 1.1molar equivalent of 2-(((N-benzyloxy)carbonyl)amino))) nicotinic acidinstead of N-((2-Pyridinylmethyloxy)carbonyl)-anthranilic acid. C₄₆ H₄₄N₆ O₈ ; MS(M+Na)⁺ =831. ¹ H NMR (methanol-d₄), δ 2.91 (m, 1H), 3.01-3.20(m, 3H), 3.5 (m, 2H), 3.9 (m, 2H), 4.6 (m, 2H), 5.0 (m, 4H), 7.1-7.3 (m,22H), 7.84 (m, 1H), 8.1 (m, 1H), 8.3 (m, 2H).

EXAMPLE 4

This example describes the synthesis of 2S,3S,5S!-2,5-bis ((N-N-(2-pyridinylmethyloxy)carbonyl!anthranyl)amino!-3-hydroxy-1,6-diphenylhexane(DD-5).

DD-5 (compound 36, Table IV) was synthesized according to the followingreaction scheme: ##STR8##

A solution of N-((2-pyridinylmethyloxy)carbonyl)-anthranilic acid (42mg, 0.15 mmol), (2S,3S,5S)-2,5-bisamino-3-hydroxyl-1,6-diphenylhexane(Stuk et al., J. Org. Chem., 59, 4040 (1994); 20 mg, 0.07 mmol), HOBT(23 mg, 0.21 mmol), TBTU (46 mg, 0.14 mmol), in DMF (10 ml) was treatedwith DIPEA (37 mg, 49 μl, 0.28 mmol) and then stirred at roomtemperature for 7 hr. The solvents were removed under vacuum, and theresidue was diluted with ethyl acetate, washed sequentially with anaqueous solution of KHSO₄ (pH=5), water, and aqueous NaHCO₃, and driedover MgSO₄. Yield: 31 mg (27%). The compound was crystallized from thesolvent system EtOAc-hexanes (1:3). The yield after crystallization was22 mg. MS (M+Na)⁺ =817; ¹ H NMR (Methanol-d₄), δ 1.77-1.81 (m, 2H),2.72-2.77 (m, 1H), 2.83-2.86 (m, 2H), 2.91 (d, J=7.7 Hz, 2H), 3.81-3.85(m, 2H), 4.61-4.70 (m, 2H), 5.19-5.21 (m, 4H), 6.99-7.05 (m, 4H),7.09-7.13 (m, 4H),7.19-7.20 (m, 2H), 7.24-7.33 (m, 8H), 7.39-7.44 (m,6H), 7.68-7.72 (m, 2H), 8.13-8.15 (m, 2H), 8.43-8.45 (m, 2H). K_(i)=74pM.

EXAMPLE 5

This example describes the synthesis of 2S,3R,4S,5S!-2- N-tert-butyloxy)carbonyl!amino!-5- ((N-N-2-pyridinylmethyloxy)carbonyl!anthranyl)amino!-3,4-dihydroxy-1,6-diphenylhexane(DN-11).

DN-11 was synthesized according to the following reaction scheme:##STR9##

A solution of(2S,3R,4S,5S)-2-amino-5-(N-benzyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane(250 mg, 0.575 mmol) and (Boc)₂ O (138 mg, 0.63 mmol) in methylenechloride (15 ml) was stirred at room temperature for 24 hr. Thevolatiles were removed under vacuum, and the residue was diluted withethyl acetate, washed sequentially with 10% solution of KHSO₄ water, andaqueous NaHCO₃, and dried over MgSO₄. The yield of product was 308 mg.The compound was crystallized from the solvent system EtOAc-hexanes 1:2.The yield of crystallized product was 270 mg (88%).

A continuous stream of hydrogen was bubbled through a solution of(2S,3R,4S,5S)-2-(N-tert-butyloxycarbonylamino)-5-(N-benzyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane(500 mg) in ethanol (80 ml) containing 10% Pd on carbon (40 mg) for 5hr(Bodanszky et al. (1984), supra, at page 153). The reaction mixture wasfiltered through celite and concentrated in vacuum to provide 316 mg of(2S,3R,4S,5S)-2-(N-tert.-butyloxycarbonylamino)-5-amino-3,4-dihydroxy-1,6-diphenylhexane(Boc-DAD).

A solution of (N-2-pyridinylmethyloxy)carbonyl)-anthranilic acid(Py-Ant, 30 mg, 0.11 mmol), Boc-DAD (40 mg, 0.1 mmol), HOBT (16 mg, 0.1mmol), TBTU (32 mg, 0.1 mmol), in DMF (20 ml) was treated with DIPEA (26mg, 35 μl, 0.2 mmol) and then stirred at room temperature for 6 hr. Thesolvents were removed under vacuum, and the residue was diluted withethyl acetate, washed sequentially with a 10% solution of KHSO₄, waterand aqueous NaHCO₃, and dried over MgSO₄. Yield: 65 mg. The compound wascrystallized from the solvent system EtOAc-hexanes (1:1). The yieldafter crystallization was 26 mg. MS(M+Na) 677; ¹ H NMR (methanol-d₄), δ1.231 (m, 9H), 2.59-2.64 (m, 2H), 2.89-2.93 (m, 1H), 2.97-3.07 (m, 2H),3.59-3.67 (m, 2H), 4.15-4.19 (m, 1H), 4.66-4.69 (m, 2H), 5.21-5.29 (m,2H), 7.06-7.09 (m, 1H), 7.11-7.14 (m, 2H), 7.17-7.25 (m, 7H), 7.30-7.32(m, 2H), 7.35-7.37 (m, 1H), 7.41-7.45 (m, 1H), 7.51-7.54 (m, 2H),7.84-7.88 (m, 1H), 8.111 (d, J=4.6 Hz, 1H), 8.51-8.52 (m, 2H). K_(i)=0.4 nM.

EXAMPLE 6

This example describes the synthesis of 2S,3R,4S,5S!-2- ((N-N-2-pyridinylmethyloxy)carbonyl !anthranyl)amino!-5- N-3-hydroxyphenyl)carbonyl!amino!-3,4-dihydroxy-1,6-diphenylhexane (ND-4).

ND-4 was synthesized according to reaction Scheme VI of Example 1.##STR10## A solution of(2S,3R,4S,5S)-2-(N-tert-butyloxycarbonyl-amino)-5-amino-3,4-dihydroxy-1,6-diphenylhexane(Boc-DAD) (40 mg, 0.1 mmol), 3-hydroxybenzoic acid (15 mg, 1.15 mmol),HOBT (16 mg, 0.1 mmol), TBTU (32 mg, 0.1 mmol), in DMF (20 ml) wastreated with DIPEA (26 mg, 35 μl, 0.2 mmol) and then stirred at roomtemperature for 4 hr. The solvents were removed under vacuum, and theresidue was diluted with ethyl acetate, washed sequentially with a 10%solution of KHSO₄, water, and aqueous NaHCO₃, and dried over MgSO₄.Yield: 42 mg (80%).

This compound (42 mg) was dissolved in TFA (3 ml) and stirred at roomtemperature for 15 min. The volatiles were removed by evaporation underreduced pressure to provide the 3-hydroxy-benzamide-substituted DADisostere as a TFA salt. The resulting compound was used in the next stepwithout further purification.

A solution of (N-2-pyridinylmethyloxy)carbonyl)-anthranilic acid(Py-Ant, 24 mg, 0.09 mmol), the above-prepared DAD isostere (0.08 mmol),HOBT (13 mg, 0.08 mmol), and TBTU (26 mg, 0.08 mmol), in DMF (20 ml) wastreated with DIPEA (30 mg, 42 μl, 0.2 mmol) and then stirred at roomtemperature for 6 hr. The solvents were removed under vacuum, and theresidue was diluted with ethyl acetate, washed sequentially with a 10%solution of KHSO₄, water, and aqueous NaHCO₃, and dried over MgSO₄.Yield: 48 mg (71%) of crude ND-4 purified by preparative TLC using thesolvent system EtOAc-hexanes (1:1).

EXAMPLE 7

This example describes the antiretroviral activity of compounds preparedin accordance with the above examples.

The inhibition constants (K_(i)) for the compounds of the above exampleswere determined using purified HIV-1 protease (wild-type, WT) (Tables I,II, IV and V) and R8Q and V82I mutant HIV-1 proteases (Table III). K_(i)is an inhibition constant of a given compound as derived by enzymekinetics. A low K_(i) represents a high affinity of the compound for theenzyme, i.e., tight binding or low dissociation. Inhibition of thecleavage was assayed using a fluorogenic substrate available fromMolecular Probes, Inc., Eugene, OR, and described in Kageyama et al.,Antimicrob. Agents. Chemother., 37, 272 (1993), and a fluorogenicsubstrate available from Bachem California, Torrance, Calif., anddescribed in Kageyama et al. (1993), supra. The inhibitory potencies ofthese compounds are set forth in Tables I-V. Percent inhibition is thepercentage inhibition of an enzyme's activity by a given compound at agiven concentration.

                                      TABLE I    __________________________________________________________________________    Influence of substituents linked to the central core on    in vitro activity.    1 #STR11##                                  %    No.       a   b   c  d  e  Y    Y'   inhib*                                       K.sub.i    __________________________________________________________________________    5. H   H   H  H  H  R--OH                             S--OH                                  38    5a.       H   OH  H  H  H  R--OH                             S--OH     3 μM    5b.       OH  H   H  H  H  R--OH                             S--OH                                  66    __________________________________________________________________________     *% inhibition at 10 μM.     Ph = phenyl.

                                      TABLE II    __________________________________________________________________________    Influence of substituents linked to the central core on    in vitro activity.    2 #STR12##                                  %    No.       a   b   c  d  e  Y    Y'   inhib*                                      K.sub.i    __________________________________________________________________________     6.       H   H   H  H  H  R--OH                             R--OH                                  55     7.       H   OH  H  H  H  R--OH                             S--OH    22 nM     8.       H   OH  H  H  H  R--OH                             R--OH    79 nM     9.       H   OH  H  H  H  S--OH                             S--OH    43 nM    10.       H   H   OH H  H  R--OH                             R--OH    330 nM       OH  H   H  H  H  R--OH                             R--OH                                  66  10 μM       H   OH  OH H  H  R--OH                             S--OH    80 nM       H   OH  H  OH H  R--OH                             S--OH    0.7 μM       H   OH  H  CH.sub.3                     H  R--OH                             S--OH    1 μM       H   OH  H  H  CH.sub.3                        R--OH                             S--OH    75 nM       H   OH  CH.sub.3                  H  H  R--OH                             S--OH    132 nM       H   NH.sub.2               H  H  H  R--OH                             S--OH    5 nM       H   OR* H  H  H  R--OH                             S--OH                                  63       H   OR**               H  H  H  R--OH                             S--OH                                   9    20.       N-2-(4-oxo-4H-1-benzopyran)                        R--OH                             S--OH                                  67       N-2-(4-hydroxyquinoline)                        R--OH                             S--OH                                  31       H   NO.sub.2               H  H  H  R--OH                             S--OH                                  40    N-3-pyridinyl--------------                        R--OH                             R--OH    0.4 μM       H   OH  H  H  H  S--OH                             H        25 nM       H   NH.sub.2               H  H  H  S--OH                             H        6 nM    __________________________________________________________________________     R* = CH.sub.2 CH.sub.2 OCH.sub.2 CH.sub.2 OCH.sub.3 ; R** = CH.sub.2     CH.sub.2 OPh.     *% inhibition at 10 μM.     Ph = phenyl.

                                      TABLE III    __________________________________________________________________________    Comparison of in vitro activity of selected compounds    against R8Q and V82I mutants and wild-type HIV(w)    2 #STR13##                                -----Ki(nM)-----    No. a b   c  d  e  Y    Y'  WT  R8Q                                       V82I    __________________________________________________________________________     7. H OH  H  H  H  R--OH                            S--OH                                22  36 51    12. H OH  OH H  H  R--OH                            S--OH                                80  40 235    15. H OH  H  H  CH.sub.3                       R--OH                            S--OH                                75  89 54    17. H NH.sub.2              H  H  H  R--OH                            S--OH                                 5  20 109    24. H OH  H  H  H  S--OH    25  34 96    __________________________________________________________________________     Ph = phenyl.

                                      TABLE IV    __________________________________________________________________________    Anthranilamide-containing C2-Symmetric inhibitors of HIV    protease.    3 #STR14##    No.  Y'   R"     W    b    V   K.sub.i (nM)    __________________________________________________________________________    26.  OH   CH.sub.3                     --   H    C   41    27.  OH   CH.sub.2 Ph                     O    H    C   2.4    28.  OH   CH.sub.2 CH.sub.3                     O    H    C   41% @ 10 μM    29.  OH   OH     CH.sub.2                          H    C   41    30.  OH   CH.sub.2 Ph                     O    --   N   3    31.  OH   CH.sub.2 Ph                     O    CH.sub.3                               C   12    32.  OH   CH.sub.2 Ph                     O    Cl   C   91    33.  OH   CH.sub.2 Ph                     O    OH   C   20    34.  OH   CH.sub.2 -2-Py                     O    H    C   0.06    35.  H    CH.sub.2 Ph                     O    H    C   1.2    36.  H    CH.sub.2 -2-Py                     O    H    C   0.07    __________________________________________________________________________     Ph = phenyl.     Py = pyridinyl.

    TABLE V      - Name Structure Formula M.W. % Inhib. Conc. K.sub.i      DN-1      ##STR15##     4  C.sub.33 H.sub.36 N.sub.2 O.sub.6 556   11.9 ±      0.6 nM                                                         DN-4      ##STR16##     5  C.sub.39 H.sub.38 N.sub.4 O.sub.7 674    1.7 ±      0.09 nM                                                         DN-5      ##STR17##     6  C.sub.39 H.sub.43 N.sub.3 O.sub.7 653   15.8 ±      1.3 nM                                                         DN-6      ##STR18##     7  C.sub.44 H.sub.47 N.sub.5 O.sub.8 773   40% @ 10 NM 15% @ 1 μM     DN-6B      ##STR19##     8  C.sub.39 H.sub.39 N.sub.5 O.sub.6 673    6.6 ±      0.3 nM                                                         DN-10      ##STR20##     9  C.sub.31 H.sub.38 N.sub.2 O.sub.6 534    3.09 ±      0.007 nM                                                          DN-11      ##STR21##     0  C.sub.37 H.sub.42 N.sub.4 O.sub.7 654    0.45 ±      0.1 nM                                                          ND-4      ##STR22##     1  C.sub.39 H.sub.38 N.sub.4 O.sub.7 674      0.7 nM      ##STR23##     2      ##STR24##     3      ##STR25##     4      ##STR26##     5

Compound 5 is a weak inhibitor of HIV PR. Compounds 5a and 5b, whichpossess 3-OH and 2-OH, respectively, show small improvements ininhibition of HIV PR. In contrast, incorporation of a hydrophilic groupinto the benzamide ring 6, in general, significantly improved inhibitorypotency. As suggested by molecular modeling studies, the 3-hydroxy and3-amino derivatives 7 and 17 (K_(i) 22 nM and 5 nM, respectively) provedto be potent inhibitors of HIV PR. Consistent with the SAR studies ofC2-symmetric diol-based inhibitors, the inhibitory potencies weredependent on the stereochemistry of the diol core (Kempf et al. (1990),supra). The 3,4 dihydroxy core possessing the R,S configuration wasobserved to be, in general, three times more potent than the corepossessing the R,R configuration, as in compounds 7 and 8. The2-methyl-5-hydroxy derivative 15, which by molecular modeling studiesappears to possess a conformation in which the aromatic ring of thebenzamide group is out of the amide plane, showed a K_(i) of 75 nM. The4-hydroxy derivative 10 showed a K_(i) of 330 nM, whereas 2-hydroxysubstitution 11 caused a dramatic loss in enzyme inhibitory potency (66%inhibition at 10 μM). The fusion of the second ring diminished bindingof inhibitors (20 and 21) to the enzyme. The C2-symmetric compoundspossessing a deshydroxy core have been shown to have improved bindingaffinities over the corresponding diols (Kempf et al. (1993), supra).Incorporation of the deshydroxy core, as in compounds 24 and 25, did notimprove the inhibitory potencies (compare 7 vs. 24 and 17 vs. 25).Compound 26 was found to be an inhibitor of HIV PR with a K_(i) of 41nM. Compound 27, which possesses a benzyloxycarbonyl substituent, was 20times more potent than compound 26, having a K_(i) of 2.4 nM.

The inhibitory potencies of selected compounds against R8Q and V82I HIVPR mutants is shown in Table III. The R8Q mutation mostly affects theP3/P3' subsite on the enzyme, whereas the V82I mutation affects theS1/S1' subsite. Tested compounds were equipotent against both thewild-type (WT) and the R8Q mutant and 0.5-4-fold less effective againstthe V82I mutant. As predicted by molecular modeling studies, the2-acylamino-benzamides, i.e., N-acyl anthranilamides, were very potent.Diol-48 (compound 34) had a K_(i) of ˜61 pM and DD-5 (compound 36) had aK_(i) of ˜74 pM.

EXAMPLE 8

This example demonstrates the anti-retroviral activity of the presentinventive compounds in CEM cells.

Using the soluble formazan assay described by Weislow et al. (J. Nat'l.Cancer Inst., 81, 577-586 (1989)), CEM cells chronically infected withHIV-1 were used to assay the anti-retroviral activity of Diol-48(compound 34) and DD-5 (compound 36). The concentration of compound thatinhibits 50% of viral activity (EC₅₀ ; determined as described inWeislow et al. (1989), supra) and the inhibition constant (K_(i), asdefined in Example 7) for the compound were determined. Diol-48 had aK_(i) of 61 pM and an EC₅₀ of 3.2×10⁻⁹ M, whereas DD-5 had a K_(i) of 74pM and an EC₅₀ of 2.4×10⁻⁹ M. These results compared favorably withanti-retroviral compounds currently undergoing clinical trials. Forexample, KNI272, ABT538, Ro31-8959, and A77003 have EC₅₀ values of4.2×10⁻⁹ M, 3.6×10⁻⁸ M (Flentge et al., 207th ACS Meeting, San Diego,Calif. (1994)), 1×10⁻⁸ M (Ho et al., J. Virol., 68, 2016 (1994), and2×10⁻⁷ (Ho et al., supra), respectively. In addition, compound DD-5showed no toxicity even at doses as high as 10⁻⁴ M and suppressed p24synthesis at concentrations ≧10⁻⁸ M. These results show that thecompounds of the present invention inhibit retroviral activity and havepotential untility as retroviral inhibitors.

EXAMPLE 9

This example describes a novel method for selective protection of one ofthe NH₂ groups of DAD, which is the intermediate for synthesis forasymmetric compounds comprising anthranilamide and benzamide subunits.

To a stirred solution of DAD (1 g, 3.3 mmol) and triethylamine (0.5 ml,3.6 mmol) in anhydrous CH₂ Cl₂ (330 ml) under nitrogen was addedbenzyloxycarbonyl chloride (Z-Cl, 510 μl, 3.6 mmol in 25 ml CH₂ Cl₂) bysyringe pump at a rate of 2.5 ml/hr. After complete addition, thestirring was continued for an additional 4 hr. The organic layer waswashed with 1% KHSO₄ (25 ml) and brine, dried over MgSO₄, and evaporatedto provide 1.34 g (92%) of crude(2S,3R,4S,5S)-2-amino-5-(N-benzyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane.The crude compound was dissolved in 25 ml ethyl acetate and treated with25 ml of petroleum ether and stored at room temperature for 12 hr. Theresulting precipitate was separated by filtration and the organic layerwas concentrated under vacuum (1.27 g; 78%; HPLC (YMS-ODS-AQ, C-18 CDCl₃: δ 2.86 (dd, J=14,0.5 Hz, 1H), 2.92 (dd, J-14,7.5 Hz, 1H), 2.94 (dd,J=14,7.5 Hz, 1H), 3.17 (dd, J=14,7.5 Hz, 1H), 355 (dd, J=10,1.5 Hz, 1H),3.70 (m, 1H), 3.82 (dd, J=10,3.5 Hz, 1H), 4.19 (ddd, J=9,7.5,1.5 Hz,1H), 4.4-4.8 (bs, 2H), 4.98 (d, J=12.5 Hz, 1H), 5.06 (d, J=12.5 Hz, 1H),6.43 (d, J=9 Hz, 1H), 7.18-7.33 (m, 15H), 7.99 (bs, 2H); MsFAB(M+H)⁺435).

All publications cited herein are hereby incorporated by reference tothe same extent as if each publication were individually andspecifically indicated to be incorporated by reference and were setforth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

What is claimed is:
 1. A method of synthesizing2S,3R,4S,5S-2-amino-5(N-benzyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane,which method comprises the steps of:(a) reacting2S,3R,4S,5S-2,5-diamino-3,4-dihydroxy-1,6-diphenylhexane andtriethylamine with benzyloxycarbonyl chloride to form crude2S,3R,4S,5S-2-amino-S-(N-benzyloxycarbonylamino)-3,4-dihydroxy-1,6-diphenylhexane;and (b) separating the reaction product.